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Abstract

Using gas chromatography mass spectrometry (GC-MS) to analyze the citric acid cycle (CAC) and related intermediates (such as glutamate, glutamine, GABA, and aspartate) is an analytical approach to identify unexpected correlations between apparently related and unrelated pathways of energy metabolism. Intermediates can be as expressed as their absolute concentrations or relative ratios by using known amounts of added reference standards to the sample. GC-MS can also distinguish between heavy labeled molecules (2H- or 13C-labeled) and the naturally occurring most abundant molecules. Applications using tracers can also assess the turnover of specific metabolic pools under various physiological and pathological conditions as well as for pathway discovery.

The following protocol is a relatively simple method that is not only sensitive for small concentrations of metabolic intermediates but can also be used in vivo or in vitro to determine the integrity of various metabolic pathways, such as flux changes within specific metabolite pools. We used this protocol to determine the role of phosphoenolpyruvate carboxykinase 1 (Pck1) gene in mouse macrophage cells to determine the percent contribution from a precursor of 13C labeled glucose into specific CAC metabolite pools.

With the development of altered gene expression in cells and mice, there is a need to understand how these deleted or over-expressed genes impact the regulation of metabolic pathways. In this protocol, we used stable isotopes to determine how the flux of glucose into the CAC altered the contribution of glucose into the pools of citrate, succinate and malate. The use of stable isotopes with targeted analysis of metabolism is just one benefit to using stable isotopes in cell culture.

The method described in this protocol for functional quantification of intracellular metabolites was done by growing bone marrow-derived macrophage cells (BMDM) in U-13C-glucose medium. The cells were extracted in organic solvent and the percent contribution of 13C glucose was calculated. The fractional amount of 13C label incorporated into each the CAC-related metabolite pools was also determined. The calculations were based on the ratio of 13C label of each intracellular metabolite versus the unlabeled metabolite; for absolute concentration analysis of cell samples, one would need to correct for reference to the intracellular volume of the extracted cells (Feldberg et al., 2009), as well as add non-interfering reference standards for quantifying of levels of CAC intermediates, as previously described (Ko et al., 2018). This protocol can also be used in isolated perfused livers or whole body metabolism studies (Yang et al., 2008a; Zhang et al., 2015).

Using a novel mouse model that had a deletion of phosphoenolpyruvate carboxykinase 1 (Pck1) in the myeloid cells (Pck1MC-KO), stable isotopes were used to determine the role of this gene in macrophages (Ko et al., 2018) with respect to glucose metabolism. The protocol explains the isolation and differentiation of BMDM. These cells were isolated, differentiated and incubated with U13C-glucose to analyze their metabolism. The BMDM cells were collected, and the fractional contribution of the precursor to the product was based on the mole percent enrichments (MPE) derived from the 13C label incorporation into the total pool of each of the metabolites (products). Mass isotopomer analysis enables the measurements of unlabeled analyte (M0) relative to the labeled analyte (M+1, 2, or 3, etc.), (Yang et al., 2008a; Kombu et al., 2011; Ko et al., 2018). The measured mass isotopomer distributions were calculated for each of the masses and expressed as mole percent enrichment (MPE) after correcting for natural isotope abundances.

LysM-specific Pck1 knock-out mice (Pck1MC-KO mice) Note: The mice were generated by crossing Pck1flox/flox mice with LysM-Cre+/− transgenic mice expressing Cre-recombinase under control of the LysM promoter. Pck1flox/flox mice were used as controls. All mice are in the C57Bl/6J background. All animals were housed in a temperature-controlled facility with a 12 h light/dark cycle in compliance with the Institutional Animal Care and Use Committee (IACUC) of Case Western Reserve University.

Isolation and differentiation of Bone Marrow-Derived Macrophages and incubation with stable isotope

Harvesting Marrow from Mouse Femur and Tibia

Prepare macrophage differentiation media (MDM) (see Recipes). Warm up the media in a 37 °C water bath for 30 min before harvesting the marrow.

Euthanize the mouse with CO2 asphyxiation and cervical dislocation and place the mouse in a beaker with 100% ethanol.

Using sterile scissors in a tissue culture hood, clip the skin and remove the skin from the lower part of the mouse body.

Remove the legs from the mouse body with scissors that have been placed in a beaker with 100% ethanol (Figure 1).

Remove all remaining hair and skin. Remove muscle, and fat surrounding the bones. Place the bones in 100% ethanol for 5 min. Pour 8 ml of MDM into a sterile 100 mm x 15 mm polystyrene Petri dish. Transfer the bones from the 100% ethanol to the Petri dish with media.

Separate at the knee joint the femoral bones from the tibia and fibula. Be careful not to break the bones.

Cut off each end of the bone as shown in Figure 1. You should have a total of 3 bones for each mouse leg in one Petri dish with media.

Figure 1. Isolating bone marrow from mouse tibia, fibula and femur. The mouse was euthanized. A. Image illustrating where to cut and remove the leg. B. Schematic diagram of the positions to be cut at mouse leg for isolating bone marrow. Cut the legs at position 1 (red dashed lines). Next remove the fur, skin and muscle from the leg. Then cut the bones at the red dotted lines to isolate the bones in the order indicated (numbers 2, 3, 4 and 5). Note that each bone is cut medially and distally.

Fill 26 G needle/1 ml syringe with macrophage differentiation media (MDM) (see Recipes). Put the tip of the needle into the bone and flush the bone marrow from both ends of the bone (Figure 2). Do this for the femur, fibula and tibia. Repeat flushing the marrow out of the bones until the bones turn white.

Figure 2. Flushing the bones. A. Image showing the marrow compartment in the bone. B and C. The femur before (B) flushing and after (C) flushing (see Video 1).

Video 1. Flushing of the bones to remove bone marrow

Collect the cells in MDM and pass them through a 70 μm cell strainer into a sterile conical tube. This removes debris that may contaminate your cells (see Video 2).

Video 2. Removing debris from isolated cells

Spin down cells at 1,950 x g for 5 min at 4 °C. Count the cells and resuspend the pellets with macrophage differentiation media (MDM) so that you can seed 5 x 106 cells and MDM into each well of the 6-well plate.

Incubate plates for 7 days at 37 °C and 5% CO2. Change media every three days with freshly made media.

Differentiation of macrophage to M1 or M2
When BMDM are almost confluent (at Day ~7), replace with new MDM. In order to polarize the macrophages, we used either lipopolysaccharide (LPS) or interleukin-4 (IL-4).

For classic M2 activation: Add interleukin-4 (IL-4) at a dose of 40 ng/ml (1.6 μl of stock solution into 4 ml of MDM) and incubate for 4 h.

Metabolism studies using U-13C Glucose

Replace the MDM in the differentiated macrophages (already treated with either LPS or IL-4) with medium that contains no glucose, 10% charcoal-stripped FBS, 100 ng/ml LPS, 5 mM U-13C glucose (99.98% enriched) and incubate overnight at 37 °C. We did this because the gene of being investigated, Pck1, is activated in the fasted state, so we removed the glucose and insulin.

At the next day, collect the media and cells. To collect media, pipet off half of the media. Keep 1 ml of the media for measurement of isotope enrichment. To collect cells, scrape the wells to dislodge the cells. Pipet up the cells and remaining media and place into conical tubes. Spin down the cells at 1,950 x g for 5 min at 4 °C and resuspend the pellets in 50 μl of 1x PBS. Save 20 μl for measurement of protein concentration by Bradford assay. Freeze sample at -20 °C to lyse cells. The protein concentrations are used for normalization in the metabolite analysis. Cells are for analysis by GC-MS. In our case, we sent the cells to the Case Western Reserve University Mouse Metabolic Phenotyping Center (MMPC) for analysis by GC-MS.

Bradford assay: Carry out the assay according to the manufacturer’s directions (Quick StartTM Bradford Protein Assay). We used 2 mg/ml BSA as a standard. Pipette 20 μl of the resuspended cells into a 1.5 ml Eppendorf tube and add 1 ml of 1x Dye reagent from the Quick StartTM Bradford Protein Assay. Mix the sample and incubate at room temperature for 5 min. Place the sample in a cuvette. Set the spectrophotometer to 595 nm. Zero the instrument with the blank sample (100 μl water and 1 ml of 1x Dye reagent). Measure the absorbance of the BSA standards and the unknown (macrophage cells). Determine the amount of protein in the sample from the standard curve (if 2 μl aliquot of sample yields a 595 nm value equivalent to 250 μg/ml of protein, then the cell sample would have a protein concentration of 2.5 mg/ml).

For the study on Pck1MC-KO macrophages, the cells were sent to the Case Western Reserve University Mouse Metabolic Phenotyping Center (MMPC) for analysis by GC-MS. The following section displays an example for the study of metabolites in the LPS-activated macrophage. The purpose of the example study was to compare the metabolites in Pck1MC-KO (experimental) and Pck1fl/fl (control) macrophages after a 24 h treatment of LPS.

Cell sample preparation
Briefly, this approach utilizes the rapid reaction of silylating reagents with alcohols, acids and amines to form silyl derivatives (Yang et al., 2008a and 2008b; Kombu et al., 2011; Zhang et al., 2015). Commercial silylating reagents are available as combinations to accelerate the reaction, as well as to react with the hindered group. For example either a mixture of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and trimethylchlorosilane (TMCS) to form a TMS derivative, or a mixture of N-Methyl-N-(t-butyldimethylsilyl)-trifluoroacetamide (MTBSTFA) and t-butyldimethylchlorosilane (TBDMS) to form TBDMS derivatives can be used (Yang et al., 2008a and 2008b; Kombu et al., 2011; Zhang et al., 2015)

For absolute quantification of analytes, as previously described (Ko et al., 2018): 50 μl of cells are spiked with selected internal reference standards such as ~5 nmole of each metabolite [13C6] citrate, [13C4] succinate, and/or [2H4]-3-hydroxyglutarate. Note: Choice of internal reference standards are dependent on precursor tracer used such that there is no interference of the M+ labeling from the tracer versus the labeled standards added; its best to use non-endogenously produced reference standards, such as tricarballylic acid.

Centrifuge the homogenate at 670 x g for 30 min at 4 °C. Decant the supernatant into a glass test tube and save on ice and process immediately Note: May freeze the supernatant at -80 °C until assaying for GC-MS.

After the GC-MS run, calculate the area under the curve for succinate, citrate and malate and the ratios to the internal standard (Table 1). After background correction, calculate the MPE% using the following formula:
MPE% = [(13C labeled M1 thru M + for each of the CAC intermediates) x (13C labeled + unlabeled) - 1] x 100 (Table 1)

Table 1. The calculation of mole percent enrichments from 13C-glucose into citrate, succinate and malate. M0, M1 to M3 were calculated based on the ratio of labeled intracellular metabolites to unlabeled and were corrected with reference to the intracellular volume of the extracted cells (Feldberg et al., 2009). The %MPE was calculated as %m2 = [(13C labeled M1 thru M + for each of the CAC intermediates) x (13C labeled + unlabeled) - 1] x 100.

Data analysis

For experimental design, the stable isotope studies are done in triplicate for each sample. The triplicates are considered an n = 1. All experiments are done for a total of n = 6 times.

MPE of M1 to M3 was calculated based on the ratio of labeled intracellular metabolites to unlabeled and corrected with reference to the intracellular volume of the extracted cells (Feldberg et al., 2009). The %MPE was calculated as MPE% = [(13C labeled M1 thru M+ for each of the CAC intermediates) x (13C labeled + unlabeled)] x 100.

For statistical analysis, we used GraphPad Prism. To compare two data sets (Pck1flox/flox and Pck1MC-KO mice), we performed a Student's t-test (Figure 7).

Figure 7. Contribution of glucose to the central metabolism of LPS-activated macrophages. BMDMs isolated from Pck1flox/flox and Pck1MC-KO mice were incubated with [U-13C] glucose in the presence of LPS for 16 h, and GC-MS was performed for the calculation of the contribution of 13C to M2-CAC. Values represent the means ± SEM for n = 6 per group. *P < 0.05 compared to Pck1flox/flox group.

Notes

Internal standards are necessary due to variation in each run. For this experiment, the samples (n = 6 per group) were all run on the GC-MS at the same time to reduce variation.

Isolation of BMDM was done for each animal one at a time. For example, one Pck1flox/flox mouse was euthanized, legs dissected and bone marrow was flushed from the femur tibia and fibula. The cells were kept cold on ice. Then the next animal was euthanized and so on. The cells were kept on ice until all of the bone marrow was collected.

The cells isolated from the bone marrow will adhere to the tissue culture plates within 24 h-48 h. The MDM contains M-CSF that will allow for myeloid cells to be differentiated into macrophages. If the cells continue to float in the media there may be contamination of the cells. If this occurs, you will need fresh media and re-isolate the bone marrow cells.

5% acetic acid in methanol/water (1:1) extraction buffer (chilled on ice)
Make methanol/water by adding 50 ml of methanol to 50 ml of dH2O to make 100 ml
In a separate graduated cylinder, combine 5 ml of acetic acid with 95 ml of the methanol/water made above

LPS stock solutions
Make up to 40 µg/µl in sterile water and aliquot
Store at -80 °C

IL-4 stock solution
Make up to 100 µg/µl in sterile water and aliquot
Store at -80 °C

Acknowledgments

The Case MMPC was supported by NIH grant U24 DK76174. The complete study on Pck1MC-KO mice has been published (Ko et al., 2018).

Competing interests

The authors declare that they have no conflicts of interest with the contents of this article.

Ethics

All animal experiments were approved by Case Western Reserve University Institutional Animal Care and Use Committee (IACUC).

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